Biological nitrogen fixation is mediated by the nitrogenase enzyme system that catalyses the ATP dependent reduction of atmospheric dinitrogen to ammonia; this reaction is the only known biological mechanism for converting nitrogen from the abundant atmospheric reservoir to a metabolically usable form. Indeed until the advent of the Haber-Bosch process, virtually all nitrogen present in the biomolecules of humans and all other organisms was fixed through the action of nitrogenase in microorganisms. Nitrogenase consists of two component metalloproteins, the MoFe-protein with the FeMo-cofactor that provides the active site for substrate reduction, and the Fe-protein that couples ATP hydrolysis to electron transfer. The mechanistic questions related to how nitrogenase overcomes the kinetic stability of the NN triple bond to fix dinitrogen under ambient conditions have intrigued chemists for the past century and serve as the focus for the present proposal. Using approaches based on biochemistry, X-ray crystallography and electron microscopy, experiments are designed to probe the nitrogenase mechanism in molecular detail. These studies will address how substrates bind to the active site FeMo-cofactor, the flow of electrons through the system, and the nucleotide dependent interactions of the nitrogenase proteins, which are essential for developing a molecular mechanism for nitrogenase function. Beyond the specific details of the reduction of dinitrogen, nitrogenase is a prototypic example of an enzyme with multiple and varied iron-sulfur clusters that participate in electron transfer and substrate reduction, as well as providing an excellent model for energy transduction of ATP hydrolysis. Consequently, these studies will further have broad implications beyond nitrogenase for diverse biochemical systems.